Methods for treating disorders that involve immunoglobulin A

ABSTRACT

Provided herein are FDC-SP polypeptides and methods of using such polypeptides. Methods include, but are not limited to, altering IgA concentration in a subject, treating a subject having signs of a disorder that includes excessive IgA production, identifying a compound that decreases the concentration of IgA in an animal, and identifying a compound that treats a condition associated with increased levels of IgA. Also provided herein is an animal that has decreased expression of an endogenous FDC-SP coding sequence. The animal may develop pathophysiological features of IgA nephropathy, and/or may display increased IgA in serum, saliva, bronchoalveolar lavage fluid, or a combination thereof; increased IgA expressing B lymphocytes in circulation, lymphoid tissue, or a combination thereof; or increased IgA production in vitro by isolated B lymphocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the §371 U.S. National Stage of InternationalApplication No. PCT/IB2012/000053, filed Jan. 17, 2012, which claims thebenefit of U.S. Provisional Application Ser. No. 61/433,449, filed Jan.17, 2011, which are incorporated by reference herein in theirentireties.

BACKGROUND

IgA nephropathy (IgAN), also known as Berger disease, is the most commoncause of chronic glomerulonephritis and leads to end stage kidneyfailure in about 25% of cases. Twenty to forty percent of IgAN patientsreceiving kidney transplants suffer disease recurrence within 5 years oftransplant. The onset of IgAN has been associated with upper respiratorytract infections that trigger the mucosal immune system. As a result ofhyper-reactivity of the mucosal immune system, B cells produce increasedamounts of IgA leading to IgA deposition in kidney glomeruli. IgAdeposition leads to glomerular inflammation, resulting in kidneydysfunction, hypertension and slow progression towards kidney failure.

Current treatments for IgAN are aimed at slowing kidney damage andinclude anti-hypertensives to control blood pressure and steroidtreatment to reduce inflammation. Long term dialysis and kidneytransplantation are used to treat end stage kidney failure but have alarge negative impact on patient quality of life. Currently, no specifictreatment is available to correct hyper-active mucosal immune responsesor reduce IgA levels.

Research into pathological mechanisms and new treatments for IgAN havebeen hampered by lack of appropriate animal models. Currently reportedmouse models for IgAN include a multigenic outbred model with variabledisease progression (ddY mouse), a single gene knockout that affects IgAdeposition but not IgA production (uteroglobulin knockout), and arecently reported mouse transgenic for a B cell activating factor (BAFFtransgenic). None of these models selectively impact on IgA productionand have to date not been widely adopted for IgAN studies.

IgAN is estimated to affect over 60,000 people in the US, and severalfold higher incidence of IgAN is reported among Asian populations. IgANis incurable and the current limited treatment options includemanagement of hypertension and administering non-specificanti-inflammatories in order to delay the need for dialysis ortransplantation. No targeted therapies for reducing IgA production areavailable.

SUMMARY OF THE INVENTION

The invention relates to the discovery and characterization ofpolypeptides that play a role in the production of IgA, and to thegeneration of an animal model that exhibits IgA nephropathy. Thecompositions and methods embodied in the present invention areparticularly useful for drug screening and/or treatment of disordersthat involve IgA.

Provided herein are uses of an FDC-SP polypeptide and a pharmaceuticallyacceptable carrier. In one embodiment, the use is in the manufacture ofa medicament for treating an IgA mediated condition. In one embodiment,the use is for treating an IgA mediated condition. An example of such acondition includes a glomerulonephritis, such as IgA nephropathy andHenoch-Schönlein purpura. Another example of such a condition includesIgA pemphigus. In one embodiment, the use is in the manufacture of amedicament for decreasing IgA concentration. In one embodiment, the useis for decreasing IgA concentration.

The FDC-SP polypeptide may include an amino acid sequence X₁X₂X₃PWX₄(SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y, F, or N,and X₄ is Y or F, and wherein the amino acid sequence of the isolatedpolypeptide has at least 90% amino acid similarity with SEQ ID NO:2 orSEQ 11) NO:4. The FDC-SP polypeptide may include an amino acid sequenceX₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y,F, or N, and X₄ is Y or F, and wherein the amino acid sequence of theisolated polypeptide has at least 90% amino acid similarity with asubset of consecutive amino acids chosen from SEQ ID NO:2 or 4.

Also provided herein are methods of using an FDC-SP polypeptide. In oneembodiment, a method includes altering IgA concentration in a subject byadministering to the subject in need thereof an effective amount of anFDC-SP polypeptide, wherein the FDC-SP polypeptide results in adecreased IgA level in the subject compared to the subject before theadministration. The IgA level may be decreased, for instance, in serum,in bronchoalveolar lavage fluid, in saliva, or a combination thereof.The method may further include identifying a subject having or at riskof an IgA mediated condition. The decrease may be a decrease of at least10%.

The FDC-SP polypeptide may include an amino acid sequence X₁X₂X₃PWX₄(SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y, F, or N,and X₄ is Y or F, and wherein the amino acid sequence of the isolatedpolypeptide has at least 90% amino acid similarity with SEQ ID NO:2 orSEQ ID NO:4. The FDC-SP polypeptide may include an amino acid sequenceX₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y,F, or N, and X₄ is Y or F, and wherein the amino acid sequence of theisolated polypeptide has at least 90% amino acid similarity with asubset of consecutive amino acids chosen from SEQ ID NO:2 or 4.

In one embodiment, a method includes treating a subject by administeringto the subject an effective amount of a FDC-SP polypeptide, wherein thesubject has signs of a disorder that includes excessive IgA production.An example of such a disorder includes a glomerulonephritis, such as IgAnephropathy and Henoch-Schönlein purpura. Another example of such adisorder includes IgA pemphigus.

Provided herein is an animal that has decreased expression of anendogenous FDC-SP coding sequence, and which develops pathophysiologicalfeatures of IgA nephropathy selected from IgA deposition in kidneys,mesangial hyperproliferation, and polypeptide deposition in glomeruli.Also provided herein is an animal that has decreased expression of anendogenous FDC-SP coding sequence, wherein the animal has at least oneof the following: increased IgA in serum, saliva, bronchoalveolar lavagefluid, or a combination thereof; increased IgA expressing B lymphocytesin circulation, lymphoid tissue, or a combination thereof; or increasedIgA production in vitro by isolated B lymphocytes; wherein the increaseis compared to a control mouse. In one embodiment, the animal may have aheterozygous disruption of an endogenous FDC-SP coding sequence. In oneembodiment, the animal may have a homozygous disruption of an endogenousFDC-SP coding sequence. In one embodiment, the animal is not a human. Inone embodiment, the animal is a mouse.

Also provided herein is a cell from the animal, wherein the cell hasdecreased expression of an endogenous FDC-SP coding sequence. Examplesof such cells include, but are not limited to, a follicular dendriticcell, a monocyte, or a macrophage. Also provided herein is a tissue fromthe animal. The tissue may be, but is not limited to, lymphoid tissue.

Provided herein are methods for identifying a compound that decreasesthe concentration of IgA in an animal. In one embodiment, the methodincludes administering to an animal a compound, and measuring theconcentration of IgA, wherein a decreased concentration of IgA in ananimal administered the compound compared to the concentration of IgAbefore the administration indicates the compound decreases theconcentration of IgA in an animal. The concentration of IgA may bedecreased, for instance, in serum, saliva, bronchoalveolar lavage fluid,or a combination thereof. The concentration of IgA may be measured bydetermining the number of IgA expressing B lymphocytes in circulation,lymphoid tissue, or a combination thereof, of the animal. A decrease inthe number of IgA expressing B lymphocytes indicates a decreasedconcentration of IgA in the animal.

Provided herein are methods for identifying a compound that treats acondition associated with increased levels of IgA. In one embodiment,the method includes administering to an animal a compound, wherein theanimal displays a sign of a condition associated with increased levelsof IgA, and evaluating a sign of a condition associated with increasedlevels of IgA, wherein a decrease in the presence of a sign indicatestreats a condition associated with increased levels of IgA. The sign maybe selected from IgA deposition in kidneys, mesangialhyperproliferation, polypeptide deposition in glomeruli, proteinurea, ora combination thereof. The animal may include decreased expression of anFDC-SP polypeptide.

Also provided herein are polypeptides. In one embodiment, a polypeptidehas immuno-modulatory activity, and includes an amino acid sequenceX₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y,F, or N, and X₄ is Y or F, and wherein the isolated polypeptide either(1) comprises no greater than 40 amino acids, or (2) comprises greaterthan 45 amino acids. In one embodiment, a polypeptide hasimmuno-modulatory activity, and includes an amino acid sequenceX₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y,F, or N, and X₄ is Y or F, and wherein the amino acid sequence of theisolated polypeptide has at least 90% amino acid and no greater than 99%similarity with SEQ ID NO:2 or SEQ ID NO:4. In one embodiment, apolypeptide has immuno-modulatory activity, and includes an amino acidsequence X₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X_(t) and X₂ are any aminoacid, X₃ is Y, F, or N, and X₄ is Y or F, and wherein the amino acidsequence of the isolated polypeptide has at least 90% similarity with asubset of consecutive amino acids chosen from SEQ ID NO:2 or 4.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers(e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide,enzyme, and protein are all included within the definition ofpolypeptide and these terms are used interchangeably. It should beunderstood that these terms do not connote a specific length of apolymer of amino acids, nor are they intended to imply or distinguishwhether the polypeptide is produced using recombinant techniques,chemical or enzymatic synthesis, or is naturally occurring. An“isolated” polypeptide is one that has been removed from a cell. Forinstance, an isolated polypeptide is a polypeptide that has been removedfrom the cytoplasm a cell, and many of the polypeptides, nucleic acids,and other cellular material of its natural environment are no longerpresent. A “purified” polypeptide is one that is at least 60% free,preferably at least 75% free, and most preferably at least 90% free fromother components of a cell.

As used herein, a polypeptide may be “structurally similar” to areference polypeptide if the amino acid sequence of the polypeptidepossesses a specified amount of sequence similarity and/or sequenceidentity compared to the reference polypeptide. Thus, a polypeptide maybe “structurally similar” to a reference polypeptide if, compared to thereference polypeptide, it possesses a sufficient level of amino acidsequence identity, amino acid sequence similarity, or a combinationthereof.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides, deoxynucleotides,peptide nucleic acids, or a combination thereof, and includes bothsingle-stranded molecules and double-stranded duplexes. A polynucleotidecan be obtained directly from a natural source, or can be prepared withthe aid of recombinant, enzymatic, or chemical techniques. Apolynucleotide described herein may be isolated. An “isolated”polynucleotide is one that has been removed from its naturalenvironment. Polynucleotides that are produced by recombinant,enzymatic, or chemical techniques are considered to be isolated andpurified by definition, since they were never present in a naturalenvironment.

A “regulatory sequence” is a nucleotide sequence that regulatesexpression of a coding sequence to which it is operably linked.Nonlimiting examples of regulatory sequences include promoters,enhancers, transcription initiation sites, translation start sites,translation stop sites, transcription terminators, and poly(A) signals.The term “operably linked” refers to a juxtaposition of components suchthat they are in a relationship permitting them to function in theirintended manner. A regulatory sequence is “operably linked” to a codingregion when it is joined in such a way that expression of the codingregion is achieved under conditions compatible with the regulatorysequence.

The term “complementary” refers to the ability of two single strandedpolynucleotides to base pair with each other, where an adenine on onepolynucleotide will base pair to a thymine or uracil on a secondpolynucleotide and a cytosine on one polynucleotide will base pair to aguanine on a second polynucleotide.

Conditions that are “suitable” for an event to occur, or “suitable”conditions are conditions that do not prevent such events fromoccurring. Thus, these conditions permit, enhance, facilitate, and/orare conducive to the event.

As used herein, an antibody that can “specifically bind” a polypeptideis an antibody that interacts only with the epitope of the antigen thatinduced the synthesis of the antibody, or interacts with a structurallyrelated epitope. An antibody that “specifically binds” to an epitopewill, under the appropriate conditions, interact with the epitope evenin the presence of a diversity of potential binding targets.

As used herein, “ex vivo” refers to a cell that has been removed fromthe body of an animal. Ex vivo cells include, for instance, primarycells (e.g., cells that have recently been removed from a subject andare capable of limited growth in tissue culture medium), and culturedcells (e.g., cells that are capable of long term culture in tissueculture medium).

As used herein, “B cell” refers to lymphocytes that are able to produceantibody that specifically bind an epitope of an antigen. Examples of Bcells include plasma B cells, memory B cells, B-1 cells, B-2 cells,marginal zone B cells, and follicular B cells. A B cell may includesurface antigens such as CD19, CD20, CD21, CD22, CD23, surfaceimmunoglobulin, Ig-alpha (also known as CD79A), and Ig-beta (also knownas CD79B).

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A, Amino acid sequence of a mouse FDC-SP polypeptide, mFDC-SP(SEQ ID NO:6), a rat FDC-SP polypeptide, rFDC-SP (SEQ ID NO:7), a humanFDC-SP polypeptide, hFDC-SP (SEQ ID NO:8), and a chimpanzee FDC-SPpolypeptide, cFDC-SP (SEQ ID NO:9). The site of cleavage of thesecretion signal is shown by the arrow. B, Nucleotide sequence of humanFDC-SP mRNA (SEQ ID NO:10).

FIG. 2. A; schematic showing strategy for constructing a deletion withinthe mouse FDC-SP gene removing a coding region encoding an FDC-SPpolypeptide (referred to a knockout or KO below). FIG. 2B. Nucleotidesequence (SEQ ID NO:14) of the gene targeting construct shown in FIG.2A. The location and sequence of the primer SC3-416 (SEQ ID NO:11), andthe location and reverse sequence of the primer SC3-412 (SEQ ID NO:12)and the primer 4R2 (SEQ ID NO:13) are shown. Also shown are the two LoxPsites.

FIG. 3. Saliva was collected from anesthetized 10-14 week old controlCD1 mice (WT) or FDC-SP transgenic (TG) mice and then animals weresacrificed by cardiac puncture to collect blood. Bronchoalveolar lavage(BALF) fluid was collected by flushing lungs with 10 mL of PBS. Levelsof IgA or IgM antibody isotypes were measured using specific ELISAassays.

FIG. 4. Saliva was collected from anesthetized 10-14 week old controlC57BL6 mice (WT) or FDC-SP knockout (KO) mice generated using thetargeting construct in FIG. 2. Animals were sacrificed by cardiacpuncture to collect blood. Bronchoalveolar lavage (BALF) fluid wascollected by flushing lungs with 10 mL of PBS. Levels of the indicatedantibody isotypes were measured using specific ELISA assays.

FIG. 5. Mesentaric lymph node, cervical lymph node, spleen, and bloodcells were collected from young adult FDC-SP KO mice and frequency ofIgA+B lymphocytes were measured by flow cytometry. Bottom panelsrepresent results from additional flow cytometry analyses which indicateotherwise normal B cell subset composition in FDC-SP KO mice. Graphsrepresent mean and SEM of 4 mice per genotype. Cervical and mesentaricrefer to lymph nodes. T1, transitional type 1, gated asB220+IgM+CD23-CD21− lymphocytes; T2, transitional type 2, gated asB220+IgM+CD23+CD21+ lymphocytes; MZ, marginal zone, gated asB220+IgM+CD23-CD21+ lymphocytes; FO, follicular or B2 cells, gated asB220+IgM+CD23+CD21− lymphocytes; pre, pre-B cells, gated asB220+IgM-CD43−lymphocytes; pro, pro-B, gated as B220+IgM-CD43+lymphocytes; Immature, immature B cells, gated as B220+IgM++CD43−lymphocytes; Mature, mature recirculating B cells, gated asB220++IgM++CD43− lymphocytes.

FIG. 6. B cells were purified from spleens of control (WT) or FDC-SP KOmice using negative selection with anti-CD43 coupled magnetic beads.Cells were cultured for 5 days with the indicated stimuli, supernatantswere harvested and IgA production was assessed by ELISA assays.

FIG. 7. B cells were purified from spleens of control or FDC-SP TG miceusing negative selection with anti-CD43 coupled magnetic beads. Cellswere cultured for 5 days with the indicated stimuli, supernatants wereharvested and IgA production was assessed by ELISA assays.

FIG. 8. B cells were purified from spleens of C57BL6 mice using negativeselection with anti-CD43 coupled magnetic beads. Cells were cultured for5 days with the indicated stimuli and the indicated percentage of asupernatant containing recombinant FDC-SP (FDC-SP SN) or a controlsupernatant (Control SN). The resulting levels of IgA production by thecultured cells were measured by ELISA assay.

FIG. 9. The indicated synthetic peptides P1-P3 corresponding to mouseFDC-SP were added at the indicated concentrations to cultures of mouse Bcells stimulated to produce IgA (TGFb1+IL-5). The resulting levels ofIgA or IgM production were assessed by ELISA assays of culturesupernatants. Percentage of cultured cells expressing IgA was alsodetermined by flow cytometry (middle graph). P1 corresponds to aminoacids 18-33 of mFDC-SP, P2 corresponds to amino acids 35-65 of mFDC-SP,and P3 corresponds to amino acids 60-84 of FDC-SP. Note that peptide P1had no effect. Control peptides C1 and C2 were scrambled versions ofcorresponding FDC-SP derived peptides. hFDC-SP, human FDC-SP; mFDC-SP,mouse FDC-SP; urn, micromolar.

FIG. 10. The effect of the indicated synthetic peptides on IgA or IgMproduction were assessed by ELISA assays of culture supernatantscollected after 5 days of culture (A) or 7 days of culture (B). Controlpeptide C5 was a scrambled version of P8. hFDC-SP, human FDC-SP;mFDC-SP, mouse FDC-SP; um, micromolar. P5 corresponds to amino acids46-59 of mFDC-SP, P7 corresponds to amino acids 68-84 of mFDC-SP, and P8corresponds to amino acids 60-65 of FDC-SP.

FIG. 11. Urine or serum collected from mice greater than one year oldwere assessed for the indicated biomarkers of kidney dysfunction.

FIG. 12. Kidneys from FDC-SP KO mice were assessed for abnormalhistology by staining sections of formalin-fixed kidney with H&E or PASstain.

FIG. 13. Kidney cryosections stained with FITC-labeled anti-IgA.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes, but is not limited to, isolatedpolypeptides having immuno-modulatory activity. A polypeptide havingimmuno-modulatory activity is referred to herein as an FDC-SPpolypeptide. An FDC-SP polypeptide is expressed by activated folliculardendritic cells (FDCs) from tonsils and TNF-α-activated FDC-like celllines, such as FDC-1 and but not by B cell lines, primary germinalcenter B cells, or anti-CD40 plus IL-4-activated B cells. FDC-SP is alsoexpressed in leukocyte-infiltrated tonsil crypts and by LPS- orStaphylococcus aureus Cowan strain 1-activated leukocytes. FDC-SP isposttranslationally modified and secreted and can bind to the surface ofB lymphoma cells, but not T lymphoma cells, and binding of FDC-SP toprimary human B cells is markedly enhanced upon activation with theT-dependent activation signals such as anti-CD40 plus IL-4 (Marshall etal., 2002, J. Immunol., 169:2381-2389).

In one embodiment, an FDC-SP polypeptide is X₁X₂X₃ProTrpX₄ (SEQ IDNO:1), wherein X₁ and X₂ are any amino acid, X₃ is Tyr, Phe, or Asn, andX₄ is Tyr or Phe. As described in Example 1, this 6-mer has been foundto have immuno-modulatory activity. In one embodiment, an FDC-SPpolypeptide includes SEQ ID NO:1. In one embodiment, an FDC-SPpolypeptide with SEQ ID NO:1 has a number of amino acids that is nogreater than any number selected from an integer between 6 and 70, i.e.,no greater than a number selected from 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, or 70 amino acids in length. In one embodiment, an FDC-SPpolypeptide with SEQ ID NO:1 has a number of amino acids that includesor is greater than any number selected from an integer between 6 and 70,i.e., includes or is greater than a number selected from 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, or 70 amino acids in length. In one embodiment, anFDC-SP polypeptide with SEQ ID NO:1 has a number of amino acids selectedfrom an integer between 6 and 70, i.e., is 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, or 70 amino acids in length.

Examples of FDC-SP polypeptides that include SEQ ID NO:1 are depicted atSEQ ID NO:2 (see amino acids 21-84 of SEQ ID NO:6 in FIG. 1, which isencoded by nucleotides 121-314 of the mRNA disclosed in the Genbankdatabase at accession number BC037156), SEQ ID NO:3 (see amino acids21-81 of SEQ ID NO:7 in FIG. 1, which is also available from the Genbankdatabase at accession number BAD77806.1), SEQ ID NO:4 (see amino acids22-85 of SEQ ID NO:8, which is also available from the Genbank databaseat accession number AAN01116, and is encoded by nucleotides 49-306 ofthe mRNA disclosed in the Genbank database at accession number AF435080[SEQ ID NO:10]), and SEQ ID NO:5 (see amino acids 22-85 of SEQ ID NO:9,which is also available from the Genbank database at accession numberXP_001160925.1). Other examples of FDC-SP polypeptides that include SEQID NO:1 are amino acids 34-65 and 60-84 of SEQ ID NO:6 and 35-64 and59-85 of SEQ ID NO:8.

Other examples of FDC-SP polypeptides of the present invention includethose that are structurally similar to the amino acid sequence of SEQ IDNO:2, 3, 4, and/or 5, or a subset of consecutive amino acids chosen fromSEQ ID NO:2, 3, 4, or 5 provided the subset includes SEQ ID NO:1. AnFDC-SP polypeptide having structural similarity with the amino acidsequence of SEQ ID NO: 2, 3, 4, and/or 5, or having structuralsimilarity with a subset of consecutive amino acids chosen from SEQ IDNO:2, 3, 4, or 5, has immuno-modulatory activity.

Structural similarity of two polypeptides can be determined by aligningthe residues of the two polypeptides (for example, a candidatepolypeptide and any appropriate reference polypeptide described herein)to optimize the number of identical amino acids along the lengths oftheir sequences; gaps in either or both sequences are permitted inmaking the alignment in order to optimize the number of identical aminoacids, although the amino acids in each sequence must nonetheless remainin their proper order. A reference polypeptide may be a polypeptidedescribed herein. In one embodiment, a reference polypeptide is afull-length FDC-SP polypeptide, such as SEQ ID NO:6, 7, 8, or 9. In oneembodiment, a reference polypeptide is an FDC-SP polypeptide that hasbeen post-translationally processed to delete the signal sequence, forinstance, SEQ ID NO:2, 3, 4, or 5. In one embodiment, a referencepolypeptide includes a subset of consecutive amino acids chosen from SEQID NO:2, 3, 4, or 5 provided the subset includes SEQ ID NO:1 (e.g.,provided the subset includes amino acids 40-45 of SEQ ID NO:2 if SEQ IDNO:2 is the reference sequence, amino acids 36-41 of SEQ ID NO:3 if SEQID NO:3 is the reference sequence, amino acids 38-43 of SEQ ID NO:4 ifSEQ ID NO:4 is the reference sequence, or amino acids 38-43 of SEQ IDNO:5 if SEQ ID NO:5 is the reference sequence. A candidate polypeptideis the polypeptide being compared to the reference polypeptide. Thus, inone embodiment, a candidate polypeptide may be between 6 and 70 aminoacids in length. A candidate polypeptide may be isolated, for example,from a cell, such as a human or mouse cell, or can be produced usingrecombinant techniques, or chemically or enzymatically synthesized. Acandidate polypeptide may be inferred from a nucleotide sequence presentin the genome of a cell.

Unless modified as otherwise described herein, a pair-wise comparisonanalysis of amino acid sequences can be carried out using the Blastpprogram of the blastp suite-2sequences search algorithm, as described byTatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), andavailable on the National Center for Biotechnology Information (NCBI)website. The default values for all blastp suite-2sequences searchparameters may be used, including general paramters: expectthreshold=10, word size=3, short queries=on; scoring parameters:matrix=BLOSUM62, gap costs=existence:11 extension:1, compositionaladjustments=conditional compositional score matrix adjustment.Alternatively, polypeptides may be compared using the BESTFIT algorithmin the GCG package (version 10.2, Madison Wis.).

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. Aconservative substitution for an amino acid in a polypeptide describedherein may be selected from other members of the class to which theamino acid belongs.

Thus, as used herein, a candidate polypeptide useful in the methodsdescribed herein includes those with at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid sequence similarity to a reference amino acid sequence.

Alternatively, as used herein, a candidate polypeptide useful in themethods described herein includes those with at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid sequence identity to the reference amino acid sequence.

In one embodiment, an FDC-SP polypeptide includes a highly chargedN-terminal region adjacent to and downstream of the secretion signal,and a moderately proline-rich central region. The highest degree ofidentity is evident in the charged N-terminal sequence. SEQ ID NO:6, 7,8, and 9 are shown in FIG. 1 in a multiple protein alignment. Identicaland conserved amino acids are marked in the consensus sequence with “!”and “*,” respectively.

In humans, FDC-SP is encoded by a single-copy coding region that maps tochromosome 4q13. The FDC-SP coding region is spread over 10 kb andcontains five exons which encode the 5′ untranslated region (exon 1),the leader peptide (exon 2), most of the N-terminal charged region (exon3), the remainder of the coding sequence (exon 4), and the 3′untranslated sequence (exon 5) (see Marshall et al., 2002, J. Immunol.,169:2381-2389). The mouse FDC-SP coding sequence maps to chromosome 5E1.An FDC-SP polypeptide may be isolated from an animal, such as a human,chimpanzee, mouse, or rat. For instance, a genomic copy of an FDC-SPcoding region may be isolated and the exon identified. An mRNA encodingan FDC-SP polypeptide, or a cDNA of such an mRNA, may be isolated. AnFDC-SP polypeptide may be produced using recombinant techniques, orchemically or enzymatically synthesized using routine methods.

The amino acid sequence of an FDC-SP polypeptide having sequencesimilarity to SEQ ID NO:2, 3, 4, and/or 5, or a subset of consecutiveamino acids chosen from SEQ ID NO:2, 3, 4, or 5 provided the subsetincludes SEQ ID NO:1, may include conservative substitutions of thecorresponding amino acids present in SEQ ID NO: 2, 3, 4, and/or 5. Aconservative substitution is typically the substitution of one aminoacid for another that is a member of the same class. For example, it iswell known in the art of protein biochemistry that an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic (such as charge, hydrophobicity, and/or hydrophilicity)may generally be substituted for another amino acid withoutsubstantially altering the secondary and/or tertiary structure of apolypeptide. For the purposes of this invention, conservative amino acidsubstitutions are defined to result from exchange of amino acidsresidues from within one of the following classes of residues: Class I:Gly, Ala, Val, Leu, and Ile (representing aliphatic side chains); ClassII: Gly, Ala, Val, Leu, Ile, Ser, and Thr (representing aliphatic andaliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr(representing hydroxyl side chains); Class IV: Cys and Met (representingsulfur-containing side chains); Class V: Glu, Asp, Asn and Gln (carboxylor amide group containing side chains); Class VI: H is, Arg and Lys(representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr,Ile, Val, Leu, Phe and Met (representing hydrophobic side chains); ClassVIII: Phe, Trp, and Tyr (representing aromatic side chains); and ClassIX: Asn and Gln (representing amide side chains). The classes are notlimited to naturally occurring amino acids, but also include artificialamino acids, such as beta or gamma amino acids and those containingnon-natural side chains, and/or other similar monomers such ashydroxyacids. SEQ ID NO:6, 7, 8, and 9 are shown in FIG. 1 in a multipleprotein alignment. Identical and conserved amino acids are marked in theconsensus sequence with “!” and “*,” respectively.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al. (1990, Science,247:1306-1310), wherein the authors indicate proteins are surprisinglytolerant of amino acid substitutions. For example, Bowie et al. disclosethat there are two main approaches for studying the tolerance of apolypeptide sequence to change. The first method relies on the processof evolution, in which mutations are either accepted or rejected bynatural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselects or screens to identify sequences that maintain functionality. Asstated by the authors, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which changes are likely to be permissive at a certain positionof the protein. For example, most buried amino acid residues requirenon-polar side chains, whereas few features of surface side chains aregenerally conserved. Other such phenotypically silent substitutions aredescribed in Bowie et al, and the references cited therein.

A polypeptide of the present invention having immunoregulatory activityinhibits IgA production by B cells in vitro. Whether a polypeptide hasimmuno-modulatory activity may be determined by in vitro assays. Anexample of an in vitro assay is described in Example 1. The assay uses Bcells, which in one embodiment may be a primary cell obtained from ananimal, such as a mouse or human, or in another embodiment may be a Bcell line. When obtained from an animal, B cells may be obtained fromlymphoid tissues such as spleen, tonsil, and lymph node. In oneembodiment, the assay includes depleting most (at least 95%) secretoryIgA⁺ (sIgA⁺) B cells, stimulating the IgA-depleted B cells with eitherLPS and IL-5 or with TGF-b1 and IL5, for 2 hours, adding an FDC-SPpolypeptide at concentrations between 0.5 micromolar and 2 micromolar tothe medium, and incubating for 5 days. Supernatants may then beharvested and tested to determine the levels of IgA. In one embodiment,a polypeptide is considered to have immuno-modulatory activity if thereis a statistically significant decrease in IgA concentration compared toa control not exposed to the FDC-SP polypeptide. In one embodiment, apolypeptide is considered to have immuno-modulatory activity if there isa decrease in IgA concentration of at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least80% compared to a control not exposed to the FDC-SP polypeptide.

In one embodiment a polypeptide of the present invention havingimmuno-modulatory activity inhibits IgA production by B cells in vivo.In one embodiment, an example of an in vivo assay is administration of apolypeptide of the present invention to a mouse, such as an FDC-SP KOmouse (see Example 1). A polypeptide is considered to haveimmuno-modulatory activity if there is a statistically significantdecrease in IgA levels in the mouse, for instance, in bronchoalveolarfluid, compared to a control mouse. In another embodiment, a polypeptideis considered to have immuno-modulatory activity if there is asignificant reduction in IgA-related kidney pathology compared to acontrol mouse.

A polypeptide of the present invention may be expressed as a fusion thatincludes an additional amino acid sequence not normally or naturallyassociated with the polypeptide. In one embodiment, the additional aminoacid sequence may be useful for purification of the fusion polypeptideby affinity chromatography. Various methods are available for theaddition of such affinity purification moieties to proteins.Representative examples include, for instance, polyhistidine-tag(His-tag) and maltose-binding protein (see, for instance, Hopp et al.(U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137),Sgarlato (U.S. Pat. No. 5,935,824), and Sharma (U.S. Pat. No.5,594,115)). In one embodiment, the additional amino acid sequence maybe a carrier polypeptide. The carrier polypeptide may be used toincrease the immunogenicity of the fusion polypeptide to increaseproduction of antibodies that specifically bind to a polypeptide of theinvention. The invention is not limited by the types of carrierpolypeptides that may be used to create fusion polypeptides. Examples ofcarrier polypeptides include, but are not limited to, keyhole limpethemacyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbitserum albumin, and the like. In another embodiment, the additional aminoacid sequence may be a fluorescent polypeptide (e.g., green, yellow,blue, or red fluorescent proteins) or other amino acid sequences thatcan be detected in a cell, for instance, a cultured cell, or a tissuesample that has been removed from an animal. If a polypeptide of thepresent invention includes an additional amino acid sequence notnormally or naturally associated with the polypeptide, the additionalamino acids are not considered when percent structural similarity to areference amino acid sequence is determined.

Polypeptides of the present invention can be produced using recombinantDNA techniques, such as an expression vector present in a cell. Suchmethods are routine and known in the art. The polypeptides may also besynthesized in vitro, e.g., by solid phase peptide synthetic methods.The solid phase peptide synthetic methods are routine and known in theart. A polypeptide produced using recombinant techniques or by solidphase peptide synthetic methods can be further purified by routinemethods, such as fractionation on immunoaffinity or ion-exchangecolumns, ethanol precipitation, reverse phase HPLC, chromatography onsilica or on an anion-exchange resin such as DEAE, chromatofocusing,SDS-PAGE, ammonium sulfate precipitation, gel filtration using, forexample, Sephadex G-75, or ligand affinity

The present invention also includes polynucleotides. In one embodiment,a polynucleotide encodes a polypeptide described herein. Also includedare the complements of such polynucleotide sequences. A person ofordinary skill can easily determine a polynucleotide sequence encoding apolypeptide described herein by reference to the standard genetic code.A polynucleotide encoding a polypeptide having immuno-modulatoryactivity is referred to herein as an FDC-SP polynucleotide. In oneembodiment, FDC-SP polynucleotides may have a nucleotide sequenceencoding a polypeptide having the amino acid sequence shown in SEQ IDNO:4. An example of the class of nucleotide sequences encoding such apolypeptide is SEQ ID NO:10. It should be understood that apolynucleotide encoding an FDC-SP polypeptide represented by SEQ ID NO:4is not limited to the nucleotide sequence disclosed at SEQ ID NO:10, butalso includes the class of polynucleotides encoding such polypeptides asa result of the degeneracy of the genetic code. For example, thenaturally occurring nucleotide sequence SEQ ID NO:10 is but one memberof the class of nucleotide sequences encoding a polypeptide having theamino acid sequence SEQ ID NO:4. The class of nucleotide sequencesencoding a selected polypeptide sequence is large but finite, and thenucleotide sequence of each member of the class may be readilydetermined by one skilled in the art by reference to the standardgenetic code, wherein different nucleotide triplets (codons) are knownto encode the same amino acid.

An FDC-SP polynucleotide of the present invention may further includeheterologous nucleotides flanking the open reading frame encoding theFDC-SP polynucleotide. Typically, heterologous nucleotides may be at the5′ end of the coding region, at the 3′ end of the coding region, or thecombination thereof. The number of heterologous nucleotides may be, forinstance, at least 10, at least 100, or at least 1000.

The present invention also includes antibodies that specifically bind apolypeptide of the present invention. In one embodiment, an antibodyspecifically binds amino acids 60-65 of SEQ ID NO:6, amino acids 56-61of SEQ ID NO:7, amino acids 59-64 of SEQ ID NO:8, and/or amino acids59-64 of SEQ ID NO:9. In one embodiment, a short polypeptide may becoupled to a carrier polypeptide to increase immunogenicity, and anadjuvant may be used to also increase immunogenicity. In one embodiment,an antibody specifically binds amino acids 34-65 of SEQ ID NO:6, aminoacids 60-84 of SEQ ID NO:6, amino acids 35-64 or SEQ ID NO:8, and/oramino acids 59-85 of SEQ ID NO:8.

Antibody may be produced using a polypeptide described herein. Theantibody may be polyclonal or monoclonal. Laboratory methods forproducing, characterizing, and optionally isolating polyclonal andmonoclonal antibodies are known in the art (see, for instance, Harlow E.et al., 1988, Antibodies: A laboratory manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor). For instance, a polypeptide ofthe present invention may be administered to an animal, such as a mammalor a chicken, in an amount effective to cause the production of antibodyspecific for the administered polypeptide. Optionally, a polypeptide maybe mixed with an adjuvant, for instance Freund's incomplete adjuvant, tostimulate the production of antibodies upon administration.

Antibody fragments include at least a portion of the variable region ofan antibody that specifically binds to its target. Examples of antibodyfragments include, for instance, scFv, Fab, F(ab′)₂, Fv, a single chainvariable region, and the like. Fragments of intact molecules can begenerated using methods well known in the art and include enzymaticdigestion and recombinant means.

An antibody of the present invention may be coupled (also referred to asconjugated) to a detectable label, e.g., a molecule that is easilydetected by various methods. Examples include, but are not limited to,radioactive elements; enzymes (such as horseradish peroxidase, alkalinephosphatase, and the like); fluorescent, phosphorescent, andchemiluminescent dyes; latex and magnetic particles; cofactors (such asbiotin); dye crystallites, gold, silver, and selenium colloidalparticles; metal chelates; coenzymes; electroactive groups;oligonucleotides, stable radicals, and others. Methods for conjugating adetectable label to antibody vary with the type of label, and suchmethods are known and routinely used by the person skilled in the art.

The present invention is also directed to compositions including one ormore polypeptides described herein. Such compositions typically includea pharmaceutically acceptable carrier. As used herein “pharmaceuticallyacceptable carrier” includes, but is not limited to, saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Additional active compounds can also beincorporated into the compositions.

A composition may be prepared by methods well known in the art ofpharmacy. In general, a composition can be formulated to be compatiblewith its intended route of administration. Administration may besystemic or local. In some aspects local administration may haveadvantages for site-specific, targeted disease management. Localtherapies may provide high, clinically effective concentrations directlyto the treatment site, with less likelihood of causing systemic sideeffects.

Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, transdermal (topical), andtransmucosal administration. In one embodiment, administration mayinclude use of a delivery tool, such as a syringe, for direct injectioninto a specific site (e.g., during surgery) or by catheter.

Solutions or suspensions can include the following components: a sterilediluent such as water for administration, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; electrolytes, such as sodium ion,chloride ion, potassium ion, calcium ion, and magnesium ion, and agentsfor the adjustment of tonicity such as sodium chloride or dextrose. pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. A composition can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic.

Compositions can include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile solutionsor dispersions. For intravenous administration, suitable carriersinclude physiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline. A composition istypically sterile and, when suitable for injectable use, should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. Prevention of the action of microorganisms can be achieved byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compound(e.g., a polypeptide described herein) in the required amount in anappropriate solvent with one or a combination of ingredients such asthose enumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle, which contains a dispersion medium and otheringredients such as from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation that may be used include vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions may include an inert diluent or an edible carrier. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,troches, or capsules. Oral compositions can also be prepared using afluid carrier. Pharmaceutically compatible binding agents can beincluded as part of the composition. The tablets, pills, capsules,troches and the like may contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the active compounds may be deliveredin the form of an aerosol spray from a pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds may be formulated intoointments, salves, gels, or creams as generally known in the art. Anexample of transdermal administration includes iontophoretic delivery tothe dermis or to other relevant tissues.

The active compounds may be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially. Liposomal suspensions can also beused as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art. Deliveryreagents such as lipids, cationic lipids, phospholipids, liposomes, andmicroencapsulation may also be used.

In one embodiment, an active compound may be associated with a targetinggroup. As used herein, a “targeting group” refers to a chemical speciesthat interacts, either directly or indirectly, with the surface of acell, for instance with a molecule present on the surface of a cell,e.g., a receptor. The interaction can be, for instance, an ionic bond, ahydrogen bond, a Van der Waals force, or a combination thereof. Examplesof targeting groups include, for instance, saccharides, polypeptides(including hormones), polynucleotides, fatty acids, and catecholamines.Another example of a targeting group is an antibody. The interactionbetween the targeting group and a molecule present on the surface of acell, e.g., a receptor, may result in the uptake of the targeting groupand associated active compound. For instance, B cell-specific antigensmay be used, such as, but not limited to, CD19, CD20, CD21, CD22, CD23,surface immunoglobulin, Ig-alpha, and Ig-beta.

When a polynucleotide is introduced into cells using any suitabletechnique, the polynucleotide may be delivered into the cells by, forexample, transfection or transduction procedures. Transfection andtransduction refer to the acquisition by a cell of new genetic materialby incorporation of added polynucleotides. Transfection can occur byphysical or chemical methods. Many transfection techniques are known tothose of ordinary skill in the art including, without limitation,calcium phosphate DNA co-precipitation, DEAE-dextrin DNA transfection,electroporation, naked plasmid adsorption, cationic liposome-mediatedtransfection (commonly known as lipofection). Transduction refers to theprocess of transferring nucleic acid into a cell using a DNA or RNAvirus.

Transgenic mice expressing increased levels of FDC-SP polypeptide showedno evidence of toxicity associated with the polypeptide, and it isexpected that administration of an FDC-SP polypeptide to an animal willnot be toxic. Toxicity and therapeutic efficacy of such active compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the ED₅₀ (the dosetherapeutically effective in 50% of the population).

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch active compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedin the methods of the invention, it may be possible to estimate thetherapeutically effective dose initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of signsand/or symptoms) as determined in cell culture. Such information can beused to more accurately determine useful doses in humans.

The compositions can be administered one or more times per day to one ormore times per week, including once every other day. The skilled artisanwill appreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with an effective amount of apolynucleotide or a polypeptide can include a single treatment or caninclude a series of treatments.

Provided herein are animals with decreased FDC-SP expression. Suchanimals exhibit pathophysiological features of IgA nephropathy includingone or more of IgA deposition in kidneys, mesangial hyperproliferation,polypeptide deposition in glomeruli, increased proteinurea, andincreased hematuria. Such animals exhibit other characteristicsincluding, but limited to, elevated IgA in serum, saliva and/orbronchoalveolar lavage fluid, and increased IgA expressing B lymphocytesin circulation and several lymphoid tissues. Specific cells of suchanimals may exhibit decreased FDC-SP expression, including, but notlimited to, follicular dendritic cells, monocytes, and/or macrophages.Animals of any species, including, but not limited to, mice, rats,rabbits, and primates, e.g., baboons, monkeys, chimpanzees, and humansmay be used to generate an animal with decreased FDC-SP expression.

In one embodiment, an animal with decreased FDC-SP expression is atransgenic animal. A “transgenic animal” is an animal containing one ormore cells bearing genetic information received, directly or indirectly,by deliberate genetic manipulation or by inheritance from a manipulatedprogenitor, such as by microinjection or infection with a recombinantviral vector. This introduced polynucleotide may be integrated within achromosome. In one embodiment, the introduced polynucleotide isintegrated into an FDC-SP locus. A transgenic animal is a non-humananimal.

In another embodiment, an animal with decreased FDC-SP expression is ananimal administered a polynucleotide, such as an siRNA, that decreasesexpression of FDC-SP.

A transgenic animal of the present invention can be broadly categorizedas a “knock-out.” A “knock-out” has a disruption in the target codingregion via the introduction of a transgene that results in a decrease offunction of the target coding region. A disruption in a target codingregion is one that has been mutated using homologous recombination orother approaches known in the art. A disrupted coding sequence can beeither a hypomorphic allele of the coding sequence or a null allele ofthe coding sequence. The term “transgene” refers to a polynucleotide,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted into the animal'sgenome in such a way as to alter the genome of the cell into which it isinserted (e.g., its insertion results in a knockout). For example, atransgene may be directed to disrupting one or more FDC-SP codingregions by homologous recombination with genomic sequences of an FDC-SPcoding region.

In one embodiment, expression of the target coding region isinsignificant or undetectable. In one embodiment, expression of thetarget coding region is decreased by at least 40%, at least 60%, atleast 80%, or at least 90% compared to a control animal. A knock-outtransgenic animal can be heterozygous or homozygous with respect to adisrupted target coding region.

A transgenic animal of the present invention includes one that carriesheterozygous or homozygous knock out in all their cells, as well asanimals which carry heterozygous or homozygous knock out in some, butnot all their cells, i.e., mosaic animals. Also provided herein arecells and tissues from a transgenic animal of the present invention.

Coding sequences may be altered in many types of animals. Targeting of acoding region involves the use of standard recombinant DNA techniques tointroduce a desired mutation into a cloned polynucleotide of a chosenlocus, e.g., a polynucleotide having a nucleotide sequence derived froman FDC-SP coding region. That mutation is then transferred throughhomologous recombination to the genome of a pluripotent, embryo-derivedstem (ES) cell. The altered stem cells are microinjected into mouseblastocysts and are incorporated into the developing mouse embryo toultimately develop into chimeric animals. In some cases, germ line cellsof the chimeric animals will be derived from the genetically altered EScells, and the mutant genotypes can be transmitted through breeding.

In order to target a coding region, the coding region of interest may becloned and modified to result in a disruption when it is inserted into atarget coding region by homologous recombination. A cloned coding regionmay be modified to include a polynucleotide encoding a selectablemarker. A selectable marker is useful for selecting stable transformantsin culture, and example include puromycin, adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolatereductase (DHFR), hygromycin-B-phosphtransferase, thymidine kinase (TK),and xanthin-guanine phosphoribosyltransferase (XGPRT). Optionally, asequence encoding a selectable marker can be flanked by recognitionsequences for a recombinase such as, e.g., Cre or Flp. For example, aselectable marker can be flanked by loxP recognition sites (34 byrecognition sites recognized by the Cre recombinase) or FRT recognitionsites such that the selectable marker can be excised from the construct(Orban, et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6861-6865, Brandand Dymecki, 2004, Dev. Cell., 6:7-28). Crossing the transgenic animalwith another animal expressing Cre or Flp can result in excision of thenucleotides between the recognition sites to result in a knock out.Thus, this technology results in the gross destruction of the codingregion of interest.

Methods for making transgenic animals are known in the art and areroutine. The transgenic animal cells of the present invention may beprepared by introducing one or more DNA molecules into a cell, which maybe a precursor pluripotent cell, such as an ES cell, or equivalent(Robertson, E. J., In: Current Communications in Molecular Biology,Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), pp. 39-44). The term “precursor” is intended to denote onlythat the pluripotent cell is a precursor to the desired (“transfected”)pluripotent cell. The pluripotent (precursor or transfected) cell can becultured in vivo in a manner known in the art (Evans, M. J. et al.,Nature 292:154-156 (1981)) to form a chimeric or transgenic animal.

An animal with decreased FDC-SP expression may be an animal administereda polynucleotide, such as an siRNA, that decreases expression of FDC-SP.Decreasing expression of an FDC-SP coding region may be accomplished byusing a portion of a polynucleotide described herein. In one embodiment,a polynucleotide for decreasing expression of an FDC-SP coding region ina cell includes one strand, referred to herein as the sense strand, ofat least 19 nucleotides, for instance, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, or 29 nucleotides (e.g., lengths useful for dsRNAi and/orantisense RNA). The sense strand is substantially identical, preferably,identical, to a target coding region or a target mRNA. As used herein,the term “identical” means the nucleotide sequence of the sense strandhas the same nucleotide sequence as a portion of the target codingregion or the target mRNA. As used herein, the term “substantiallyidentical” means the sequence of the sense strand differs from thesequence of a target mRNA at least 1%, 2%, 3%, 4%, or 5% of thenucleotides, and the remaining nucleotides are identical to the sequenceof the mRNA.

In one embodiment, a polynucleotide for decreasing expression of anFDC-SP coding region in a cell includes one strand, referred to hereinas the antisense strand. The antisense strand may be at least 19nucleotides, for instance, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29nucleotides. In one embodiment, a polynucleotide for decreasingexpression of an FDC-SP coding region in a cell includes substantiallyall of a coding region, or in some cases, an entire coding region. Anantisense strand is substantially complementary, preferably,complementary, to a target coding region or a target mRNA. As usedherein, the term “substantially complementary” means that at least 1%,2%, 3%, 4%, or 5% of the nucleotides of the antisense strand are notcomplementary to a nucleotide sequence of a target coding region or atarget mRNA.

The present invention includes methods for using the polypeptidesdisclosed herein. In one embodiment, the methods include administeringto a subject an effective amount of a polypeptide described herein. Thesubject can be, for instance, a member of the family Muridae (a murineanimal such as rat or mouse), or a primate, such as a human.

In one embodiment, the methods may include decreasing IgA concentrationin a subject. In this aspect of the invention, an “effective amount” isan amount effective to result in a decrease of in the IgA concentrationin a subject. Without intending to be limited by theory, the method mayresult in inhibition of IgA production by the subject's B cells, and/orthe method may result in inhibition of generation of IgA producing Bcells.

The decrease of IgA concentration may be in any tissue or fluid of thesubject. For example, an IgA concentration may be decreased in thesubject's serum, bronchoalveolar lavage fluid, saliva, gut, or acombination thereof. The decrease in IgA concentration does not requirea decrease in all tissues and fluids of a subject. For instance, the IgAconcentration may decrease in a subject's serum but not in the subject'ssaliva. In one embodiment, the decrease in IgA concentration may be adecrease of at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, or at least 80% compared to theIgA concentration in the same tissue or fluid of the subject before theadministration. Methods for determining the concentration of IgA in atissue or fluid are known in the art and routine.

The present invention also includes methods of treating certain diseasesin a subject. The subject may be a mammal, including members of thefamily Muridae (a murine animal such as rat or mouse), or a primate,such as a human. As used herein, the term “disease” refers to anydeviation from or interruption of the normal structure or function of apart, organ, or system, or combination thereof, of a subject that ismanifested by a characteristic sign or set of signs. As used herein, theterm “sign” refers to objective evidence of a disease present in asubject. Signs associated with diseases referred to herein and theevaluation of such signs is routine and known in the art. Diseasesinclude conditions associated with excessive IgA production. Examples ofsuch conditions include, but are not limited to, IgA nephropathy,Henoch-Schönlein purpura, and IgA pemphigus. Typically, whether asubject has a disease, and whether a subject is responding to treatment,may be determined by evaluation of signs associated with the disease.For instance, signs of IgA nephropathy include hematuria, elevatedlevels of circulating IgA-fibronectin complex, and granular depositionof IgA and C3 in a widened renal mesangium, with foci of segmentalproliferative or necrotizing lesions.

Treatment of a disease can be prophylactic or, alternatively, can beinitiated after the development of a disease. Treatment that isprophylactic, for instance, initiated before a subject manifests signsof a disease, is referred to herein as treatment of a subject that is“at risk” of developing a disease. An example of a subject that is atrisk of developing a disease is a person having a risk factor. Riskfactors for one disease, IgA nephropathy, are thought to includeactivation of mucosal defenses by, for instance, immunization, andgeographical location, where IgA nephropathy is the most commonglomerular disease in the Far East (including China, Japan, and SouthKorea) and Southeast Asia (including Philippines, Singapore, andThailand). Treatment can be performed before, during, or after theoccurrence of the diseases described herein. Treatment initiated afterthe development of a disease may result in decreasing the severity ofthe signs of the disease, or completely removing the signs.

In some aspects, the methods typically include administering to thesubject an effective amount of an FDC-SP polypeptide, The subject mayhave symptoms of a disease that includes excessive IgA production. Asused herein, an “effective amount” is an amount effective to inhibitdecrease IgA levels in a subject, decrease signs associated with adisease, or the combination thereof. Whether a polypeptide is expectedto function in methods of the present invention relating to treatmentcan be evaluated using the animal model described herein.

Another method of the present invention includes contacting a cell witha polypeptide described herein. The cell may be ex vivo. The cell may bea B cell, a T cell, or dendritic cell. Examples of B cells includesplasma B cells, memory B cells, B-2 cells, marginal zone B cells, andfollicular B cells. The cells may be animal cells, such as vertebratecells, including murine (rat or mouse), or primate cells, such as humancells. In one embodiment the cells may be obtained from, for instance,spleen, primary lymph node, secondary lymph node, or plasma. Otherexamples of animals from which cells may be obtained include atransgenic animal that expresses increased amounts of FDC-SP and ananimal described herein that expresses decreased amounts of FDC-SP. Inone embodiment, cell lines may be used. Examples of such cell lines mayinclude FDC-1 cells, BK cells, or L cells containing an FDC-SPexpression vector or induced to express FDC-SP using TNF-alpha. In oneembodiment, such as when the cells are B cells, the cells are firstinduced with either LPS and IL-5 or with TGF-b1 and IL5. In oneembodiment, for instance when the cell is a B cell, the contacting mayresult in decreasing IgA production by the B cells, and/or inhibition ofgeneration of IgA producing B cells.

The methods of the present invention can include administering to asubject having a disease or at risk of developing a disease acomposition including an effective amount of a polypeptide of thepresent invention, wherein the concentration of IgA in a tissue and/orfluid is decreased, a sign associated with the disease is decreased, ora combination thereof. Methods for administering a polypeptide of thepresent invention include, but are not limited to, oral, respiratory,and/or parenteral administration.

The polynucleotides of the present invention can also be administered toa subject in combination with other therapeutic compounds to increasethe overall therapeutic effect. Therapeutic compounds useful for thetreatment of the diseases described herein are known and used routinely.Agents for treating diseases described herein are available that may beused as a second, supplemental agent, to complement the activity of thepolypeptides described herein. Such agents may include, for instance,steroids.

Also included in the present invention are methods for identifying acompound that decreases the production of IgA by a cell. In oneembodiment the method includes contacting a cell, such as a B cell, witha compound, incubating the cell and the agent under conditions suitablefor culturing the cell, and measuring the production of IgA by the cell.The B cell may be stimulated to produce IgA prior to the contacting, orthe B cell may already be producing IgA. For instance, the B cell mayconstitutively produce IgA or it may be isolated from an animal as anIgA producing B cell. The cell used in the method may be an ex vivocell, such as a cell line or a cell removed from an animal. The animalmay be a wild type animal, or the animal disclosed herein (an animalproducing increased amounts of IgA). The cell contacted with the agenthaving decreased IgA production when compared to IgA production by acorresponding control cell that was not contacted with the compoundindicates the compound decreases the production of IgA by the cell. Thecompound can be, but is not limited to, a chemical compound, including,for instance, an organic compound, an inorganic compound, a metal, apolypeptide, a non-ribosomal polypeptide, a polyketide, or apeptidomimetic compound. The sources for potential compounds to bescreened include, for instance, chemical compound libraries, cellextracts of plants and other vegetations.

The present invention also includes methods for using the animal modeldisclosed herein. As set forth in the Examples, an animal expressingdecreased levels of FDC-SP has a phenotype that is characterized bypathophysiological features of IgA nephropathy including one or more ofIgA deposition in kidneys, mesangial hyperproliferation, polypeptidedeposition in glomeruli, increased proteinurea, and increased hematuria.Such a phenotype is similar to the signs associated with IgA nephropathyin humans.

Thus, the present invention provides a model system that includes theanimals disclosed herein, and methods useful in the study of aspects ofthe etiology of diseases associated with increased levels of IgA, suchas IgA nephropathy. The methods are also useful for screening andselecting for compounds that have an effect on diseases associated withincreased levels of IgA, such as IgA nephropathy, the further study ofthese compounds, and the possible administration of selected compoundsto humans in order to regulate diseases associated with increased levelsof IgA, such as IgA nephropathy.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1

We have established that mice deficient in the immuno-modulatory peptideFDC-SP develop the features of IgA nephropathy (IgAN). FDC-SP KO micedevelop up to 10 fold elevation of IgA in serum, saliva andbronchoalveolar lavage fluid. IgA expressing B lymphocytes aresignificantly increased in circulation and several lymphoid tissues andisolated B lymphocytes show enhanced IgA production in vitro. These miceshow evidence of IgA deposition in kidneys and moderate kidneypathology, characterized by mesangial hyperproliferation and proteindeposition in some glomeruli. Serum creatinine and urea levels arewithin normal levels consistent with chronic kidney dysfunction ratherthan acute severe injury.

Materials and Methods

FDC-SP-Deficient and Transgenic Mice

FDC-SP-deficient mice were generated by Biogen-Idec using the targetingconstruct illustrated in FIG. 2. The construct was linearized andtransfected into 129/Sv ES cells by electroporation. G418 resistantcolonies were screened by PCR and Southern blot and a correctly targetedclone was injected into C57BL6 blastocysts, which were then implantedinto pseudo-pregnant foster mothers. Chimeric offspring were identifiedby coat color and bred to assess germline transmission. Pups positivefor the targeted FDC-SP allele were crossed with Cre transgenics togenerate the collapsed targeted FDC-SP locus. The collapsed locus wastransmitted in the germline through subsequent back-crossing to C57BL6mice, during which the Cre transgene was removed. HomozygousFDC-SP-deficient mice were generated after back-crossing to C57BL6 for 7generations at University of Manitoba. Mice were genotyped using DNAextracted from ear punches, using the following primers. The wild typeallele was amplified with GGGATAAAGTGATAAAAACGAATAGCCA (SEQ ID NO:11)and ACGGAAATCCAGAAGATGCAAGCCT (SEQ ID NO:12) to result in a 430 bpproduct. The knockout allele was amplified withGGGATAAAGTGATAAAAACGAATAGCCA (SEQ ID NO:11) and GGAGGAGTAGAAGGTGGCGCGAAG(SEQ ID NO:13) to result in a 522 bp product.

FDC-SP transgenic CD1 mice (i.e., mice constitutively expressing FDC-SPin lymphoid tissues) were generated as described (Al-Alwan et al., 2007,J. Immunol., 178:7859-7867). All experimental animals were housed at theCentral Animal Care Facility (University of Manitoba, Winnipeg, MB) incompliance with the guidelines established by the Canadian Council onAnimal Care.

Flow Cytometry Analyses

Single cell suspensions were generated from the indicated tissues andwere pre-incubated with hybridoma supernatant containing Fc receptorblocking antibody. The indicated conjugated Abs were added from thefollowing panel: V500-labeled anti-CD4, PE anti-CD5, FITC anti-CD11b,FITC anti-CD21, PE anti-CD23, PerCP anti-CD45R/B220, FITCanti-CD54/B220, PE anti-CD43, PE anti-Gr1 and APC anti-IgM (all BDBiosciences), Pacific Blue anti-CD8, AlexaFluor647 anti-mouse CD4 and PEanti-mouse IgA (eBioscience). Stained cells were washed and acquired ona FACS Canto II flow cytometer (BD Biosciences). Data were analyzedusing FlowJo software (TreeStar).

Immunization and Antibody Measurements

Serum, saliva and bronchoalveolar lavage fluid were collected from 10-14week old FDC-SP-deficient mice, FDC-SP transgenic mice and strain, ageand sex-matched controls. Where indicated mice were immunizedintraperitoneally with NP-OVA, alum and LPS. For ELISA assays,ninety-six well assay plates were coated overnight at 4° C. with captureantibodies or antigen diluted in carbonate coating buffer (0.015MNa₂CO₃, 0.035M NaHCO₃, 0.05% NaN₃, pH 9.6). Total antibodies levels weredetermined by coating with anti-mouse IgM or IgA (Jackson ImmunoResearchLaboratories) and NP-specific antibodies levels were determined bycoating plates with NP20-BSA (Biosearch Technologies). Detection wascarried out using biotinylated anti-mouse IgM or IgA antibodies(Southern Biotechnology) followed by streptavidin alkaline phosphatase.

B Cell Isolation and Cultures

Mouse splenocytes were collected and B cells were purified by negativeselection with the CD43 MicroBeads and MACS columns (Miltenyi Biotech).Purified B cells were incubated in dishes coated with goat anti-mouseIgA (4 μg/ml) for 70 min at 4° C., resulting in >95% depletion of sIgA⁺cells. IgA-depleted B cells were then washed and resuspended in completemedium (RPMI 1640 containing penicillin-streptomycin, 2-mercaptoethanoland 10% FBS). A total of 4 10⁵ cells/well were cultured inflat-bottomed, 96-well tissue-culture plates in a volume of 100 μlcomplete medium containing 10 μg/ml LPS (Escherichia coli 0127:B8; SigmaChemical Co.), 100 U/ml murine IL-5 (R&D Systems) and 1 ng/ml TGF-β1(R&D Systems). After 2 hours of culture an additional 100 μl/well ofmedium containing the indicated FDC-SP peptides was added. After 5 daysof culture, supernatants were harvested for ELISA analysis and cells forflow cytometry. FDC-SP containing L cell supernatants were generatedfrom stable transfectants generated as described (Al-Alwan et al., 2007,J. Immunol., 178:7859-7867). Serum-free supernatants were prepared byextensively washing and culturing confluent L cells in medium containing0.5% BSA overnight. Supernatants were harvested and used immediately oraliquoted and stored frozen at −80° C. Synthetic FDC-SP peptides werepurchased from Neo BioScience (Cambridge, Mass.) and were greater than98% pure.

Urine and Kidney Analyses:

Kidneys were harvested from 8 month old FDC-SP deficient mice and eitherembedded in O.C.T. compound (Tissue Tek) and snap frozen using liquidnitrogen for cryosectioning or formalin fixed for pathology analyses.Cryosections were cut at 8 μm using a cryostat and placed onto slides(Fisherbrand Superfrost/Plus). Frozen sections were fixed for 15-20minutes using ice cold acetone, blocked with 10% normal goat serum for 1hr and stained with FITC anti-IgA (Southern Biotechnology). Aftermounting, sections were imaged using a confocal microscope (UltraviewLCI, Perkin-Elmer). For pathology analyses, formalin fixed kidneys wereparaffin embedded, sectioned and stained with hematoxylin and eosin orperiodic acid—Schiff stains. For 24 hour urine protein assay, individualmice were put in a metabolic cage with water but no food for 24 hours.Collected urine was clarified by centrifugation at 3000 rpm and proteinmeasured using Bradford protein assay (Biorad). Total protein wasdetermined by multiplying protein concentration by volume and wasindependently measured 3 times per animal.

Results

FDC-SP-Deficient (KO) Mice and FDC-SP Transgenic (TG) Mice

FIG. 1A shows an alignment of FDC-SP protein sequences from mouse, rat,human and chimpanzee. FIG. 1B shows the mouse FDC-SP nucleotide sequence(cDNA). Transgenic mice constitutively expressing FDC-SP in all lymphoidtissues were generated as described in (Al-Alwan et al., 2007, J.Immunol., 178:7859-7867). FIG. 2A illustrates the gene targetingconstruct used to generate mice devoid of FDC-SP (FDC-SP KO). FIG. 2Brepresents the nucleotide sequence of the FDC-SP gene targetingconstruct.

FDC-SP Regulates IgA Production In Vivo

As shown in FIG. 3, saliva was collected from anesthetized 10-14 weekold control CD1 mice (WT) or FDC-SP transgenic (TG) mice and thenanimals were sacrificed by cardiac puncture to collect blood.Bronchoalveolar lavage (BALF) fluid was collected by flushing lungs with10 mL of PBS. Levels of IgA or IgM antibody isotypes were measured usingspecific ELISA assays. The results show FDC-SP transgenic mice havereduced IgA levels in serum, bronchoalveolar lavage and saliva, whereasIgM levels are not reduced.

As shown in FIG. 4, saliva was collected from anesthetized 10-14 weekold control C57BL6 mice (WT) or FDC-SP knockout (KO) mice and thenanimals were sacrificed by cardiac puncture to collect blood.Bronchoalveolar lavage (BALF) fluid was collected by flushing lungs with10 mL of PBS. Levels of the indicated antibody isotypes were measuredusing specific ELISA assays. The results show FDC-SP knockout mice haveenhanced IgA levels that persist for over one year.

Together the data in FIGS. 3 and 4 indicate that the products of theFDC-SP gene can regulate IgA production in vivo.

FDC-SP is not Required for B Lymphocyte Development, but Controls theGeneration of IgA+Cells

As shown in FIG. 5, lymph node, spleen and blood cells were collectedfrom young adult FDC-SP KO mice and frequency of IgA+B lymphocytes weremeasured by flow cytometry. Bottom panels represent results fromadditional flow cytometry analyses which indicate otherwise normal Bcell subset composition in FDC-SP KO mice. Graphs represent mean and SEMof 4 mice per genotype. The results show that FDC-SP KO mice haverelatively normal B lymphocyte populations but an increased frequency ofIgA+B cells.

FDC-SP Derived Peptides can Directly Suppress B Cell IgA Production InVitro

As shown in FIG. 6, B cells were purified from spleens of control (WT)or FDC-SP KO mice using negative selection with anti-CD43 coupledmagnetic beads. Cells were cultured for 5 days with the indicatedstimuli, supernatants were harvested and IgA production was assessed byELISA assays. The results show that B cells isolated from FDC-SPknockout mice generate more IgA when stimulated in vitro.

As shown in FIG. 7, B cells were purified from spleens of control orFDC-SP TG mice using negative selection with anti-CD43 coupled magneticbeads. Cells were cultured for 5 days with the indicated stimuli,supernatants were harvested and IgA production was assessed by ELISAassays. The results show B cells isolated from FDC-SP transgenic micegenerate less IgA when stimulated in vitro.

As shown in FIG. 8, B cells were purified from spleens of C57BL6 miceusing negative selection with anti-CD43 coupled magnetic beads. Cellswere cultured for 5 days with the indicated stimuli and the indicatedpercentage of a supernatant containing recombinant FDC-SP (FDC-SP SN) ora control supernatant (Control SN). IgA production was assessed by ELISAassays. The results show that addition of recombinant FDC-SP in the formof supernatant of L cells transfected with FDC-SP expression vectorsuppressed IgA production in vitro.

As shown in FIG. 9, the indicated synthetic peptides P1-P3 correspondingto mouse FDC-SP were purchased and added to cultures of mouse B cellsstimulated to produce IgA. The resulting levels of IgA or IgM productionwere assessed by ELISA assays of culture supernatants. Percentage ofcultured cells expressing IgA was also determined by flow cytometry(middle graph). Note that peptide P1 had no effect. Control peptides C1and C2 are scrambled versions of corresponding FDC-SP derived peptides.The results show portions of FDC-SP have direct inhibitory activity on Bcell IgA production in vitro.

As shown in FIG. 10, the effect of the indicated synthetic peptides onIgA or IgM production were assessed by ELISA assays of culturesupernatants. Control peptide C5 is a scrambled version of P8. Theresults show the 6-mer represented by P8 is sufficient to inhibit B cellIgA production in vitro.

Urine and Kidney Analyses Show IgA Nephropathy-Like Disease in FDC-SP KOMice

As shown in FIG. 11, urine or serum collected from mice greater than oneyear old were assessed for the indicated biomarkers of kidneydysfunction. The results show significant proteinurea and hematuria inFDC-SP KO mice, indicating chronic nephropathy.

As shown in FIG. 12, kidneys from FDC-SP KO mice were assessed forabnormal histology by staining sections of formalin-fixed kidney withH&E or PAS stain. Abnormal glomeruli with mesangial hypercellularitywere noted and glomerular capillary hyaline thrombi were present,indicating protein deposits.

As showed in FIG. 13, further analysis of kidney cryosections stainedwith FITC-labeled anti-IgA showed evidence of mesangial IgA deposition.

Together these results indicate that the elevated IgA levels in FDC-SPKO mice result in kidney pathology similar to that of IgA nephropathy.

The FDC-SP mouse model offers a tool to dissect the molecular andcellular pathology of IgAN and test new targeted treatments. Theavailable evidence suggests that FDC-SP acts to regulate induction orretention of IgA producing B cells at mucosal sites. The model thusreplicates a key aspect of the human disease etiology, where disruptionof mucosal B cell regulation is thought to lead to abnormal systemic IgAproduction. FDC-SP is not normally expressed in kidney tissue, thus thismodel also facilitates studies focused on the pathology attributable todysregulated IgA.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A method for altering IgA concentration in asubject comprising administering to the subject in need thereof aneffective amount of an FDC-SP polypeptide, wherein the FDC-SPpolypeptide results in a decreased IgA level in the subject compared tothe subject before the administration, wherein the subject is a human,and wherein the subject has signs of a disorder that comprises excessiveIgA production, wherein the disorder is glomerulonephritis, IgApemphigus, or a combination thereof, wherein the glomerulonephritis isIgA nephropathy, Henoch-Schönlein purpura, or a combination thereof. 2.The method of claim 1 wherein the IgA level is decreased in serum, inbronchoalveolar lavage fluid, in saliva, or a combination thereof. 3.The method of claim 1 wherein the method further comprises identifying asubject having or at risk of an IgA mediated condition.
 4. The method ofclaim 1 wherein the FDC-SP polypeptide comprises an amino acid sequenceX₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y,F, or N, and X₄ is Y or F, and wherein the amino acid sequence of theisolated polypeptide has at least 80% amino acid similarity with SEQ IDNO:2 or SEQ ID NO:4.
 5. The method of claim 1 wherein the FDC-SPpolypeptide comprises an amino acid sequence X₁X₂X₃PWX₄ (SEQ ID NO:1),wherein X₁ and X₂ are any amino acid, X₃ is Y, F, or N, and X₄ is Y orF, and wherein the amino acid sequence of the isolated polypeptide hasat least 80% amino acid similarity with a subset of consecutive aminoacids chosen from SEQ ID NO:2 or
 4. 6. The method of claim 1 wherein thedecrease is a decrease of at least 10% compared to the IgA level in thesame tissue or fluid of the subject before the administration.
 7. Amethod for treating a subject comprising: administering to the subjectan effective amount of an FDC-SP polypeptide, wherein the subject hassigns of a disorder that comprises excessive IgA production, wherein thedisorder comprises a glomerulonephritis, IgA pemphigus, or a combinationthereof, wherein the subject is a human.
 8. The method of claim 7wherein the glomerulonephritis is selected from IgA nephropathy andHenoch-Schönlein purpura.
 9. A method for treating a subject comprising:administering to the subject an effective amount of an FDC-SPpolypeptide, wherein the subject has signs of a disorder that comprisesexcessive IgA production, wherein the disorder comprises IgA pemphigus.10. The method of claim 1 wherein the FDC-SP polypeptide comprises anamino acid sequence X₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are anyamino acid, X₃ is Y, F, or N, and X₄ is Y or F, and wherein the aminoacid sequence of the isolated polypeptide comprises no greater than 31amino acids and has at least 80% amino acid similarity with amino acids35-64 of SEQ ID NO:8.
 11. The method of claim 1 wherein the polypeptidecomprises an amino acid sequence X₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁and X₂ are any amino acid, X₃ is Y, F, or N, and X₄ is Y or F, andwherein the isolated polypeptide comprises no greater than 25 aminoacids and has at least 80% amino acid similarity with amino acids 59-85of SEQ ID NO:8.
 12. The method of claim 7 wherein the FDC-SP polypeptidecomprises an amino acid sequence X₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁and X₂ are any amino acid, X₃ is Y, F, or N, and X₄ is Y or F.
 13. Themethod of claim 7 wherein the FDC-SP polypeptide comprises an amino acidsequence X₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid,X₃ is Y, F, or N, and X₄ is Y or F, and wherein the amino acid sequenceof the isolated polypeptide has at least 80% amino acid similarity withSEQ ID NO:2 or SEQ ID NO:4.
 14. The method of claim 7 wherein the FDC-SPpolypeptide comprises an amino acid sequence X₁X₂X₃PWX₄ (SEQ ID NO:1),wherein X₁ and X₂ are any amino acid, X₃ is Y, F, or N, and X₄ is Y orF, and wherein the amino acid sequence of the isolated polypeptide hasat least 80% amino acid similarity with a subset of consecutive aminoacids chosen from SEQ ID NO:2 or
 4. 15. The method of claim 7 whereinthe FDC-SP polypeptide comprises an amino acid sequence X₁X₂X₃PWX₄ (SEQID NO:1), wherein X₁ and X₂ are any amino acid, X₃ is Y, F, or N, and X₄is Y or F, and wherein the amino acid sequence of the isolatedpolypeptide comprises no greater than 31 amino acids and has at least80% amino acid similarity with amino acids 35-64 of SEQ ID NO:8.
 16. Themethod of claim 7 wherein the polypeptide comprises an amino acidsequence X₁X₂X₃PWX₄ (SEQ ID NO:1), wherein X₁ and X₂ are any amino acid,X₃ is Y, F, or N, and X₄ is Y or F, and wherein the isolated polypeptidecomprises no greater than 25 amino acids and has at least 80% amino acidsimilarity with amino acids 59-85 of SEQ ID NO:8.
 17. The method ofclaim 7 wherein the administering results in a decreased IgA level inthe subject compared to the subject before the administering.
 18. Themethod of claim 17 wherein the decreased IgA level is a decrease of atleast 10% compared to the IgA concentration in the same tissue or fluidof the subject before the administration.
 19. The method of claim 17wherein the IgA level is decreased in serum, in bronchoalveolar lavagefluid, in saliva, or a combination thereof.